Project Summary Living cells have a complex and often precise organization in space and time. Understanding how proteins and other biomolecules form functional networks in vivo is a major goal of modern biology, and paramount to understanding both their normal functions as well as dysfunctions. Cryo-electron tomography (cryo-ET) is currently the only imaging technique that provides 3D information about biological structures inside cells, at a resolution better than ~100 , and up to ~30 with subtomogram averaging. However, even 30 resolution does not provide the detail needed to identify most proteins or visualize their interactions with one another. Therefore, we will develop a new hybrid approach and software tool, TYGRESS (Tomography Guided 3D Reconstruction of Subcellular Structures), to improve the resolution of cellular cryo-electron microscopy (cryo- EM) to ~15 , i.e., a level at which protein domains can be recognized and compared to known atomic structures. Our approach combines the complementary strengths of two imaging techniques: cryo- ET/subtomogram averaging and cryo-EM single-particle reconstruction. While the final TYGRESS average is a single-particle reconstruction with the benefit of a higher resolution than possible by cryo-ET, cryo-tomograms serve as essential reference frames for particle picking and alignment, steps that are not otherwise possible for projection images of complex cellular specimens. We will build the infrastructure to make this new tool available to a wide scientific community. We will also apply this new tool to study the structure and function of three important and dynamic assemblies inside cells: (i) The molecular organization and function of cilia and flagella, (ii) a molecular movie of rotavirus entry and host cell infection, and (iii) the molecular mechanisms by which proteins are transported across or inserted into the endoplasmic reticulum (ER) membrane. TYGRESS will provide an essential and innovative bridge between high-resolution structures of isolated biomolecules (x- ray, NMR, single-particle cryo-EM) and visualization of intracellular dynamics by live-cell fluorescence imaging.